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Tumor-associated macrophages (TAMs) constitute a subset of nonneoplastic cells in tumor stroma and influence cancer progression in solid tumors. The clinical significance of TAMs in urinary bladder cancer(UBC) is controversial.

Methods

We prospectively studied 103 patients with stage pT1–T4 UBC treated with cystectomy and pelvic lymph node dissection. Tumor sections were immunostained with M2-specific macrophage marker CD163 and proliferation marker Ki-67. The expression of these markers in cancer cells as well as macrophage infiltration (MI) in tumor stroma was analyzed in relation to clinical data and outcome.

M2-specific MI tends to be inversely correlated with LN metastasis and improved CSS in UBC. MI might have protective impact in CD163-positive tumors. Expression of CD163 in cancer cells is significantly correlated with MI and might have a tumor promoting impact.

OBJECTIVES: To investigate the possibility of detecting sentinel lymph nodes (SNs) in patients with urinary bladder cancer (BCa) intra-operatively and whether the histopathological status of the identified SNs reflected that of the lymphatic field.

PATIENTS AND METHODS: We studied 103 patients with BCa pathological stage T1-T4 who were treated with cystectomy and pelvic lymph node (LN) dissection during 2005-2011 at the Department of Urology, Linköping University Hospital. Radioactive tracer Nanocoll 70 MBq and blue dye were injected into the bladder wall around the primary tumour before surgery. SNs were detected ex vivo during the operation with a handheld Geiger probe (Gamma Detection System; Neoprobe Corp., Dublin, OH, USA). All LNs were formalin-fixed, sectioned three times, mounted on slides and stained with haematoxylin and eosin. An experienced uropathologist evaluated the slides.

RESULTS: The mean age of the patients was 69 years, and 80 (77%) were male. Pathological staging was T1-12 (12%), T2-20 (19%), T3-48 (47%) and T4-23 (22%). A mean (range) number of 31 (7-68) nodes per patient were examined, totalling 3 253 nodes. LN metastases were found in 41 patients (40%). SNs were detected in 83 of the 103 patients (80%). Sensitivity and specificity for detecting metastatic disease by SN biopsy (SNB) varied between LN stations, with average values of 67% and 90%, respectively. LN metastatic density (LNMD) had a significant prognostic impact; a value of ≥8% was significantly related to shorter survival. Lymphovascular invasion (LVI) occurred in 65% of patients (n = 67) and was significantly associated with shorter cancer-specific survival (P < 0.001).

CONCLUSION: We conclude that SNB is not a reliable technique for peri-operative localization of LN metastases during cystectomy for BCa; however, LNMD has a significant prognostic value in BCa and may be useful in the clinical context and in BCa oncological and surgical research. LVI was also found to be a prognostic factor.

Copper (Cu) is essential for living organisms, and acts as a cofactor in many metabolic enzymes. To avoid the toxicity of free Cu, organisms have specific transport systems that 'chaperone' the metal to targets. Cancer progression is associated with increased cellular Cu concentrations, whereby proliferative immortality, angiogenesis and metastasis are cancer hallmarks with defined requirements for Cu. The aim of this study is to gather all known Cu-binding proteins and reveal their putative involvement in cancers using the available database resources of RNA transcript levels. Using the database along with manual curation, we identified a total of 54 Cu-binding proteins (named the human Cu proteome). Next, we retrieved RNA expression levels in cancer versus normal tissues from the TCGA database for the human Cu proteome in 18 cancer types, and noted an intricate pattern of up- and downregulation of the genes in different cancers. Hierarchical clustering in combination with bioinformatics and functional genomics analyses allowed for the prediction of cancer-related Cu-binding proteins; these were specifically inspected for the breast cancer data. Finally, for the Cu chaperone ATOX1, which is the only Cu-binding protein proposed to have transcription factor activities, we validated its predicted over-expression in patient breast cancer tissue at the protein level. This collection of Cu-binding proteins, with RNA expression patterns in different cancers, will serve as an excellent resource for mechanistic-molecular studies of Cu-dependent processes in cancer.

Rectal cancer is treated with preoperative radiotherapy (RT) to downstage the tumor, reduce local recurrence, and improve patient survival. Still, the treatment outcome varies significantly and new biomarkers are desired. Collagen I (Col-I) is a potential biomarker, which can be visualized label-free by second harmonic generation (SHG). Here, we used SHG to identify Col-I changes induced by RT in surgical tissue, with the aim to evaluate the clinical significance of RT-induced Col-I changes. First, we established a procedure for quantitative evaluation of Col-I by SHG in CDX2-stained tissue sections. Next, we evaluated Col-I properties in material from 31 non-RT and 29 RT rectal cancer patients. We discovered that the Col-I intensity and anisotropy were higher in the tumor invasive margin than in the inner tumor and normal mucosa, and RT increased and decreased the intensity in inner tumor and normal mucosa, respectively. Furthermore, higher Col-I intensity in the inner tumor was related to increased distant recurrence in the non-RT group but to longer survival in the RT group. In conclusion, we present a new application of SHG for quantitative analysis of Col-I in surgical material, and the first data suggest Col-I intensity as a putative prognostic biomarker in rectal cancer. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License.

The recent years have been characterised by a rapid development of whole slide imaging (WSI) especially in its applications to histology. The application of WSI technology to cytology is less common because of technological problems related to the three-dimensional nature of cytology preparations (which requires capturing of z-stack information, with an increase in file size and usability issues in viewing cytological preparations). The aim of this study is to provide a review of the literature on the use of digital cytology and provide an overview of cytological applications of WSI in current practice as well as identifying areas for future development.

Context: Within digital pathology, digitalization of the grossing procedure has been relatively underexplored in comparison to digitalization of pathology slides.

Aims: Our investigation focuses on the interaction design of an augmented reality gross pathology workstation and refining the interface so that information and visualizations are easily recorded and displayed in a thoughtful view.

Settings and Design: The work in this project occurred in two phases: the first phase focused on implementation of an augmented reality grossing workstation prototype while the second phase focused on the implementation of an incremental prototype in parallel with a deeper design study.

Subjects and Methods: Our research institute focused on an experimental and “designerly” approach to create a digital gross pathology prototype as opposed to focusing on developing a system for immediate clinical deployment.

Statistical Analysis Used: Evaluation has not been limited to user tests and interviews, but rather key insights were uncovered through design methods such as “rapid ethnography” and “conversation with materials”.

Results: We developed an augmented reality enhanced digital grossing station prototype to assist pathology technicians in capturing data during examination. The prototype uses a magnetically tracked scalpel to annotate planned cuts and dimensions onto photographs taken of the work surface. This article focuses on the use of qualitative design methods to evaluate and refine the prototype. Our aims were to build on the strengths of the prototype's technology, improve the ergonomics of the digital/physical workstation by considering numerous alternative design directions, and to consider the effects of digitalization on personnel and the pathology diagnostics information flow from a wider perspective. A proposed interface design allows the pathology technician to place images in relation to its orientation, annotate directly on the image, and create linked information.

Conclusions: The augmented reality magnetically tracked scalpel reduces tool switching though limitations in today's augmented reality technology fall short of creating an ideal immersive workflow by requiring the use of a monitor. While this technology catches up, we recommend focusing efforts on enabling the easy creation of layered, complex reports, linking, and viewing information across systems. Reflecting upon our results, we argue for digitalization to focus not only on how to record increasing amounts of data but also how these data can be accessed in a more thoughtful way that draws upon the expertise and creativity of pathology professionals using the systems.

To provide guidance during stereotactic biopsy in brain tumors, fluorescence spectroscopy was used in ten patients. It was shown that the fiber optical probe could provide real-time guidance with clear fluorescence in all patients.

A fiber optic probe was developed for guidance during stereotactic brain biopsy procedures to target tumor tissue and reduce the risk of hemorrhage. The probe was connected to a setup for the measurement of 5-aminolevulinic acid (5-ALA) induced fluorescence and microvascular blood flow. Along three stereotactic trajectories, fluorescence (n = 109) and laser Doppler flowmetry (LDF) (n = 144) measurements were done in millimeter increments. The recorded signals were compared to histopathology and radiology images. The median ratio of protoporphyrin IX (PpIX) fluorescence and autofluorescence (AF) in the tumor was considerably higher than the marginal zone (17.3 vs 0.9). The blood flow showed two high spots (3%) in total. The proposed setup allows simultaneous and real-time detection of tumor tissue and microvascular blood flow for tracking the vessels.

BACKGROUND: The aim of this study was to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC)cell lines in an in vivo xenograft model, and to identify treatment-induced changes in the EGFR signaling pathway that could be used as markersfor cetuximab treatment response.

METHODS: The in vitro cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UTSCC-45, was assessed using a crystal violet assay. In order to determine the corresponding in vivo sensitivity, UT-SCC-14 and UT-SCC-45 xenografts were generated in female BALB/c (nu/nu) nude mice. Mice were given three injections of intraperitoneal cetuximab or PBS and the tumor volume was recorded continuously. The expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki67 was investigated by immunohistochemistry.

RESULTS: The treatment sensitive UT-SCC-14 cells were found to have an intrinsic cetuximab sensitivity (ICmabS) of 0.15 whereas the ICmabS of the insensitive cell line UT-SCC-45 was 0.78. The corresponding size ratio between untreated and cetuximab treated xenografts was 0.22 and 0.83 for UT-SCC-14 and UT-SCC-45, respectively. UT-SCC-14 cells had a higher baseline expression of pEGFR as compared to UT-SCC-45. Furthermore, in UT-SCC-14 xenografts there was a decrease in EGFR, pEGFR and pSrc upon cetuximab treatment. In contrast, a slight cetuximab-induced increase in EGFR, pEGFR and pSrc was observed in treatment-resistant UT-SCC-45 xenografts.

CONCLUSIONS: The in vitro treatment sensitivity was reproduced in the in vivo model and cetuximab sensitivity was found to associate with a treatment-induced reduction in pEGFR and pSrc.

Cell fusion is a natural biological process in normal development and tissue regeneration. Fusion between cancer cells and macrophages results in hybrids that acquire genetic and phenotypic characteristics from both maternal cells. There is a growing body of in vitro and in vivo data indicating that this process also occurs in solid tumors and may play a significant role in tumor progression. However, investigations of the response of macrophage: cancer cell hybrids to radiotherapy have been lacking. In this study, macrophage: MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation, both hybrids and their maternal MCF-7 cells were treated with 0 Gy, 2.5 Gy and 5 Gy.-radiation and examined by clonogenic survival and comet assays at three time points (0 h, 24 h, and 48 h). Compared to maternal MCF-7 cells, the hybrids showed increased survival fraction and plating efficiency (colony formation ability) after radiation. The hybrids developed less DNA-damage, expressed significantly lower residual DNA-damage, and after higher radiation dose showed less heterogeneity in DNA-damage compared to their maternal MCF-7 cells. To our knowledge this is the first study that demonstrates that macrophage: cancer cell fusion generates a subpopulation of radioresistant cells with enhanced DNA-repair capacity. These findings provide new insight into how the cell fusion process may contribute to clonal expansion and tumor heterogeneity. Furthermore, our results provide support for cell fusion as a mechanism behind the development of radioresistance and tumor recurrence.

The oral thrombin inhibitor ximelagatran was withdrawn in the late clinical trial phase because it adversely affected the liver. In approximately 8% of treated patients, drug-induced liver injury (DILI) was expressed as transient alanine transaminase (ALT) elevations. No evidence of DILI had been revealed in the pre-clinical in vivo studies. A whole genome scan study performed on the clinical study material identified a strong genetic association between the major histocompatibility complex alleles for human leucocyte antigens (HLA) (HLA-DR7 and HLA-DQ2) and elevated ALT levels in treated patients. An immunemediated pathogenesis was suggested. Here, we evaluated whether HLA transgenic mice models could be used to investigate whether the expression of relevant HLA molecules was enough to reproduce the DILI effects in humans. In silico modelling performed in this study revealed association of both ximelagatran (pro-drug) and melagatran (active drug) to the antigen-presenting groove of the homology modelled HLA-DR7 molecule suggesting "altered repertoire" as a key initiating event driving development of DILI in humans. Transgenic mouse strains (tgms) expressing HLA of serotype HLA-DR7 (HLA-DRB1*0701, -DRA*0102), and HLA-DQ2 (HLA-DQB1*0202, -DQA1*0201) were created. These two lines were crossed with a human (h) CD4 transgenic line, generating the two tgms DR7xhCD4 and DQ2xhCD4. To investigate whether the DILI effects observed in humans could be reproduced in tgms, the mice were treated for 28 days with ximelagatran. Results revealed no signs of DILI when biomarkers for liver toxicity were measured and histopathology was evaluated. In the ximelagatran case, presence of relevant HLA-expression in a preclinical model did not fulfil the prerequisite for reproducing DILI observed in patients. Nonetheless, for the first time an HLA-transgenic mouse model has been investigated for use in HLA-associated DILI induced by a low molecular weight compound. This study shows that mimicking of genetic susceptibility, expressed as DILI-associated HLA-types in mice, is not sufficient for reproducing the complex pathogenesis leading to DILI in man.

It is possible to estimate hepatic triglyceride content by calculating the proton density fat fraction (PDFF), using proton magnetic resonance spectroscopy (less thansuperscriptgreater than1less than/superscriptgreater thanH-MRS), instead of collecting and analyzing liver biopsies to detect steatosis. However, the current PDFF cut-off value (5%) used to define steatosis by magnetic resonance was derived from studies that did not use histopathology as the reference standard. We performed a prospective study to determine the accuracy of less thansuperscriptgreater than1less than/superscriptgreater thanH-MRS PDFF in measurement of steatosis using histopathology analysis as the standard. We collected clinical, serologic, less thansuperscriptgreater than1less than/superscriptgreater thanH-MRS PDFF, and liver biopsy data from 94 adult patients with increased levels of liver enzymes (6 months or more) referred to the Department of Gastroenterology and Hepatology at Linköping University Hospital in Sweden from 2007 through 2014. Steatosis was graded using the conventional histopathology method and fat content was quantified in biopsy samples using stereological point counts (SPCs). We correlated less thansuperscriptgreater than1less than/superscriptgreater thanH-MRS PDFF findings with SPCs (r = 0.92; P less than.001). less thansuperscriptgreater than1less than/superscriptgreater thanH-MRS PDFF results correlated with histopathology results (ρ = 0.87; P less than.001), and SPCs correlated with histopathology results (ρ = 0.88; P less than.001). All 25 subjects with PDFF values of 5.0% or more had steatosis based on histopathology findings (100% specificity for PDFF). However, of 69 subjects with PDFF values below 5.0% (negative result), 22 were determined to have steatosis based on histopathology findings (53% sensitivity for PDFF). Reducing the PDFF cut-off value to 3.0% identified patients with steatosis with 100% specificity and 79% sensitivity; a PDFF cut-off value of 2.0% identified patients with steatosis with 94% specificity and 87% sensitivity. These findings might be used to improve non-invasive detection of steatosis.

From experiments in mice in which the prostaglandin E2 (PGE2) synthesizing enzyme mPGES-1 was genetically deleted, as well as from experiments in which PGE2 was injected directly into the brain, PGE2 has been implicated as a mediator of inflammatory induced anorexia. Here we aimed at examining which PGE2 receptor (EP1–4) that was critical for the anorexic response to peripherally injected interleukin-1β (IL-1β). However, deletion of neither EP receptor in mice, either globally (for EP1, EP2, and EP3) or selectively in the nervous system (EP4), had any effect on the IL-1β induced anorexia. Because these mice were all on a C57BL/6 background, whereas previous observations demonstrating a role for induced PGE2 in IL-1β evoked anorexia had been carried out on mice on a DBA/1 background, we examined the anorexic response to IL-1β in mice with deletion of mPGES-1 on a C57BL/6 background and a DBA/1 background, respectively. We confirmed previous findings that mPGES-1 knock-out mice on a DBA/1 background displayed attenuated anorexia to IL-1β; however, mice on a C57BL/6 background showed the same profound anorexia as wild type mice when carrying deletion of mPGES-1, while displaying almost normal food intake after pretreatment with a cyclooxygenase-2 inhibitor. We conclude that the involvement of induced PGE2 in IL-1β evoked anorexia is strain dependent and we suggest that different routes that probably involve distinct prostanoids exist by which inflammatory stimuli may evoke an anorexic response and that these routes may be of different importance in different strains of mice.

Malignant melanoma might develop from melanocytic nevi in which the growth-arrested state has been broken. We analyzed the gene expression of young and senescent human melanocytes in culture and compared the gene expression data with a dataset from nevi and melanomas. A concordant altered gene expression was identified in 84 genes when comparing the growth-arrested samples with proliferating samples. TUBB3, which encodes the microtubule protein tubulin β-3, showed a decreased expression in senescent melanocytes and nevi and was selected for further studies. Depletion of tubulin β-3 caused accumulation of cells in the G2/M phase and decreased proliferation and migration. Immunohistochemical assessment of tubulin β-3 in benign lesions revealed strong staining in the superficial part of the intradermal components, which faded with depth. In contrast, primary melanomas exhibited staining without gradient in a disordered pattern and strong staining of the invasive front. Our results describe an approach to find clinically useful diagnostic biomarkers to more precisely identify cutaneous malignant melanoma and present tubulin β-3 as a candidate marker. This article is protected by copyright. All rights reserved.

Type I IFN and nucleic acid-sensing TLRs are both strongly implicated in the pathogenesis of lupus, with most patients expressing IFN-induced genes in peripheral blood cells and with TLRs promoting type I IFNs and autoreactive B cells. About a third of systemic lupus erythematosus patients, however, lack the IFN signature, suggesting the possibility of type I IFN-independent mechanisms. In this study, we examined the role of type I IFN and TLR trafficking and signaling in xenobiotic systemic mercury-induced autoimmunity (HgIA). Strikingly, autoantibody production in HgIA was not dependent on the type I IFN receptor even in NZB mice that require type I IFN signaling for spontaneous disease, but was dependent on the endosomal TLR transporter UNC93B1 and the endosomal proton transporter, solute carrier family 15, member 4. HgIA also required the adaptor protein-3 complex, which transports TLRs from the early endosome to the late endolysosomal compartments. Examination of TLR signaling pathways implicated the canonical NF-kappa B pathway and the proinflammatory cytokine IL-6 in autoantibody production, but not IFN regulatory factor 7. These findings identify HgIA as a novel type I IFN-independent model of systemic autoimmunity and implicate TLR-mediated NF-kappa B proinflammatory signaling from the late endocytic pathway compartments in autoantibody generation.

Visualization of the tumor is crucial for differentiating malignant tissue from healthy brain during surgery, especially in the tumor marginal zone. The aim of the study was to introduce a fluorescence spectroscopy-based hand-held probe (HHF-probe) for tumor identification in combination with the fluorescence guided resection surgical microscope (FGR-microscope), and evaluate them in terms of diagnostic performance and practical aspects of fluorescence detection.

Material and Methods

Eighteen operations were performed on 16 patients with suspected high-grade glioma. The HHF-probe and the FGR-microscope were used for detection of protoporphyrin (PpIX) fluorescence induced by 5-aminolevulinic acid (5-ALA) and evaluated against histopathological analysis and visual grading done through the FGR-microscope by the surgeon. A ratio of PpIX fluorescence intensity to the autofluorescence intensity (fluorescence ratio) was used to quantify the spectra detected by the probe.

Results

Fluorescence ratio medians (range 0 – 40) measured by the probe were related to the intensity of the fluorescence in the FGR-microscope, categorized as “none” (0.3, n = 131), “weak” (1.6, n = 34) and “strong” (5.4, n = 28). Of 131 “none” points in the FGR-microscope, 88 (67%) exhibited fluorescence with the HHF-probe. For the tumor marginal zone, the area under the receiver operator characteristics (ROC) curve was 0.49 for the FGR-microscope and 0.65 for the HHF-probe.

Conclusions

The probe was integrated in the established routine of tumor resection using the FGR-microscope. The HHF-probe was superior to the FGR-microscope in sensitivity; it detected tumor remnants after debulking under the FGR-microscope. The combination of the HHF-probe and the FGR-microscope was beneficial especially in the tumor marginal zone.

Neuroinflammation plays an influential role in Alzheimers disease (AD), although the mechanisms underlying this phenomenon remain largely unknown. Microglia are thought to be responsible for the majority of these effects and can be characterized into resting (M0), proinflammatory (M1), or anti-inflammatory (M2) functional phenotypes. We investigated the effects of conditioned macrophage media, as an analogue to microglia, on the transfer of oligomeric amyloid beta (oA beta) between differentiated SH-SY5Y cells. We also investigated how the different inflammatory environments related to intercellular and intracellular changes. We demonstrate that M2 products decrease interneuronal transfer of oA beta, while recombinant interleukin (IL)-4, IL-10, and IL-13 increase transfer. There were no alterations to the mRNA of a number of AD-related genes in response to the combination of oA beta and M0, M1, or M2, but several intracellular proteins, some relating to protein trafficking and the endosomal/lysosomal system, were altered. Stimulating microglia to an M2 phenotype may thus slow down the progression of AD and could be a target for future therapies. (C) 2017 Elsevier Inc. All rights reserved.

The current treatment recommendation for T2-3N0M0 glottic squamous cell carcinoma (SCC) in the Nordic countries comprises of radiotherapy (RT) and chemoradiotherapy (CRT). Tumor radiosensitivity varies and another option is primary surgical treatment, which underlines the need for predictive markers in this patient population. The aim of the present study was to investigate the relation of the proteins WRAP53 beta, survivin and p16INK4a to RT/CRT response and ultimate outcome of patients with T2-T3N0 glottic SCC. Protein expression was determined using immunohistochemistry on tumors from 149 patients consecutively treated with RT or CRT at Helsinki University Hospital, Karolinska University Hospital, and Linkping University Hospital during 1999-2010. Our results demonstrate a significantly better 5-year relapse-free survival, disease-free survival (DFS), disease-specific survival and overall survival of patients with T3N0 tumors treated with CRT compared with RT alone. Patients with tumors showing a cytoplasmic staining of WRAP53 beta revealed significantly worse DFS compared with those with nuclear staining. For survivin, we observed a trend towards better 5-year DFS in patients with strong nuclear survivin expression compared with those with weak nuclear survivin expression (p=0.091). Eleven (7%) tumors showed p16 positivity, with predilection to younger patients, and this age group of patients with p16-positive SCC had a significantly better DFS compared with patients with p16-negative SCC. Taken together, our results highlight WRAP53 beta as a potential biomarker for predicting RT/CRT response in T2-T3N0 glottic SCC. p16 may identify a small but distinct group of glottic SCC with favorable outcome. Furthermore, for T3N0 patients better outcome was observed following CRT compared to RT alone.